Improved Anti-Leukemic Pre-Clinical Efficacy of a Protac Based MDM2 Degrader in a Large AML Cohort

Mouse double minute 2 homolog (MDM2) is a E3 ubiquitin ligase that promotes p53 tumor suppressor degradation. Many tumors harbor mutations in TP53 that don't bind MDM2, nevertheless, MDM2 has emerged as a therapeutic target in the treatment of TP53 wild-type cancers. Although in acute myeloid leukemia (AML) incidence of TP53 mutations is low (15-20%) inactivation of wild-type p53 protein frequently occurs through overexpression of its negative regulator MDM2. Thus MDM2 is an attractive target for novel therapeutics in AML tailored to treat TP53-wild type AML overexpressing MDM2. Several MDM2 inhibitors are currently in clinical trials. However, p53 stabilization upregulates MDM2, which limits the clinical efficacy of the inhibitors. Proteolysis Targeting Chimeric (PROTAC) molecules recruit targeted proteins for degradation and therefore can improve efficacy by removing the targeted protein. We developed a MDM2 degrader, MI-265, that binds to MDM2 and targets it for degradation thus removing the repression of p53, which results in apoptosis induction in leukemic cells. We tested MI-265 in a panel of 96 primary AML samples in ex-vivo cultures. AML blood and bone marrow samples were collected after obtaining University of Michigan IRB approved informed consent from AML patients at Michigan Medicine. Leukemia stem cells (LSC) were enriched by positive selection with CD34 antibody. Cells were plated in LSC conducive medium containing growth factors. Cell death and apoptosis induction was measured by flow-cytometry for DAPI-Annexin V binding after incubation with MDM2-inhibitor and MDM2-degrader for 3 days in the presence of drug. The AML cells were obtained from newly diagnosed AML (N=82) or relapse/refractory AML (N=14). The median IC50s was 120 pM (range 32 pm to 23 nM). In contrast median IC50 for the inhibitor MI-1061 was 20,000 fold higher at 2.3 µM (range 3 nM to 455 µM). Sensitivity and resistance to MDM2-degrader and inhibitor were similar in most of the samples suggesting that the mechanism of action of both drugs are also similar. The AML cells were heterogeneous for the expression of mutant FLT-3 (36%), NPM1 (22%), IDH1/2 (16%), CEBPalpha (2%), DNMT3A (2%) and other mutations (10%). Based on classical karyotype 30% of them had a normal karyotype and 55% had abnormal karyotype (15% data missing). The MDM2-degrader resistant LSCs (IC50 greater than 1 µM) were analyzed for TP53 mutation status and 8 of 14 were found to harbor mutations in TP53. The degradation of MDM2 and overexpression of p53 was confirmed by immunoblotting after a 4 hr exposure of LSCs to degrader. Moreover, treatment of normal CD34 positive hematopoietic cells in apoptosis assay and colony formation assays suggests lack of toxicity to normal hematopoiesis. In conclusion, MI-265 represents a highly potent and efficacious MDM2 degrader, and is currently undergoing extensive preclinical evaluation for the treatment of acute leukemias.